An ex vivo model of interactions between extracellular vesicles and peripheral mononuclear blood cells in whole blood

Abstract Extracellular vesicles (EVs) can be loaded with therapeutic cargo and engineered for retention by specific body sites; therefore, they have great potential for targeted delivery of biomolecules to treat diseases. However, the pharmacokinetics and biodistribution of EVs in large animals remain relatively unknown, especially in primates. We recently reported that when cell culture‐derived EVs are administered intravenously to Macaca nemestrina (pig‐tailed macaques), they differentially associate with specific subsets of peripheral blood mononuclear cells (PBMCs). More than 60% of CD20+ B cells were observed to associate with EVs for up to 1 h post‐intravenous administration. To investigate these associations further, we developed an ex vivo model of whole blood collected from healthy pig‐tailed macaques. Using this ex vivo system, we found that labelled EVs preferentially associate with B cells in whole blood at levels similar to those detected in vivo. This study demonstrates that ex vivo blood can be used to study EV‐blood cell interactions.

Recent studies have begun to uncover the pharmacokinetics and biodistribution of EVs in larger animals, including in nonhuman primates (Driedonks et al., 2022;Haney et al., 2022).We recently reported that when EVs isolated from cell culture are administered intravenously to Macaca nemestrina (pig-tailed macaques), they differentially associate with specific subsets of peripheral blood mononuclear cells (PBMCs) (Driedonks et al., 2022).Intriguingly, flow cytometry identified CD20 + B cells as the predominant immune cell population capable of interacting with EVs, and this finding was consistent for EVs that were labelled genetically (with GFP) and post-production with a self-quenching lipid dye.
In this study, we sought to establish an ex vivo model to further interrogate EV-blood cell associations.We reasoned that an ex vivo model would have several advantages to in vivo studies, including precise control of experimental conditions; minimal invasiveness, that is, only one donor intervention (single blood draw) rather than administration and repeated sampling; and applicability to donors of multiple species, including humans.Flow cytometry and nanoluciferase assays tracked the distribution of labelled EVs in the plasma, PBMCs, and red blood cell compartments of ex vivo blood.Here, we show that EVs derived from both Expi293F and U-87 MG cells disseminate to all blood compartments but appear to interact preferentially with CD20 + B cells.This study demonstrates that ex vivo blood can be used to study EV-blood cell interactions recapitulating in vivo results.
For MemGlow labeling, U-87 EVs were adjusted to 1E+10 particles/mL in PBS and stained with 200 nM MemGlow 700 nm at RT for 30 min, protected from light.Excess dye was removed by ultrafiltration as above, and labelled EVs were stored overnight at 4˚C prior to whole blood experiments.

. EV quantification by nano flow cytometry
Particle concentration, size, and %GFP positivity of EV preparations were measured with a NFCM Flow NanoAnalyzer (NanoFCM Co., Ltd) following the manufacturer's instructions and as previously reported (Arab et al., 2021).Briefly, lasers were aligned and calibrated separately for particle concentration using fluorescent 250 nm silica nanoparticles at a concentration of 2.19E+10 (NanoFCM, catalog #QS2503) and for size using a premixed silica nanosphere cocktail containing monodisperse nanoparticle populations of 68 nm, 91 nm, 113 nm, and 155 nm in diameter (NanoFCM, catalog #516 M-Exo).DPBS was used as the blank for background correction.For quantification of EVs using NFCM, MemGlow labelled and unlabelled PalmGRET-EV preparations were diluted 50-fold or 10,000-fold in DPBS, respectively, and U-87-derived EVs were diluted 100-fold in DPBS.
Particle signal acquisition was performed for 1 min at constant pressure of 1 kPa at an event rate between 1,500 and 10,000 events/min.The side-scattering signal and fluorescent signal of each sample were calculated using the NanoFCM Professional Suite V2.0 software.

. EV spike-in experiments with ex vivo blood
Blood was obtained from donor pigtailed macaques (Macaca nemestrina) in procedures approved by the Johns Hopkins University Animal Care and Use Committee (ACUC) and conducted following the Weatherall Report, the Guide for the Care and Use of Laboratory Animals, and the USDA Animal Welfare Act.Up to 20 mL of whole blood per donor was drawn by venipuncture into a sterile 50-mL Luer-lock syringe tube (BD catalog #309653) containing acid citrate dextrose (ACD) at an ACD-to-whole blood ratio of 1:5.Blood was processed within 1 h of collection.Under sterile conditions in a cell culture hood, whole blood was transferred to a separate 50 mL conical tube and further aliquoted into sterile 2-mL or 5-mL Eppendorf tubes.For time-course experiments, 1 mL of whole blood was incubated with 8E+08 MemGlow 700-labeled PalmGRET-EVs or an equal volume of DPBS for up to 24 h in an incubated rotator at 37˚C, with the "Mix" mode selected at a speed of 8 rpm (Benchmark Scientific Roto-Therm Plus Incubated Rotator, item #H2024).Dose-response assays were conducted by incubating 4 mL of whole blood with 1.4E+08, 8E+08, 4E+09, or 1.8E+10 PalmGRET-EVs for 30 min.Dosages were established to (1) roughly match those used in our previous in vivo studies; (2) allow for detection of signal above background after processing; and (3) avoid the input exceeding 10% of total volume.One tube containing whole blood incubated with DPBS of volume equal to 2E+10 EVs served as the treatment control group.Tubes were removed from the rotating incubator at each timepoint, and 100 µL of blood was set aside for flow cytometry analysis.The remaining blood was processed as described below for nanoluciferase activity assays.

. Fractionation of EV-treated whole blood
Different blood compartments (plasma, PBMCs, and RBCs) were separated by density gradient centrifugation using SepMate-15 tubes (STEMCELL Technologies, catalog #85415) to reduce blood processing time and user variability, as described previously (Russell et al., 2022).Each whole blood sample was diluted 1:1 in PBS-EDTA (1 mM) + 2% FBS buffer (PBS-EDTA-FBS buffer) in a 15-mL conical tube and mixed well.Diluted blood was overlaid carefully on SepMate-15 tubes each containing up to 4 mL Lymphoprep (STEMCELL Technologies, catalog #07851).The layered blood was centrifuged at 1,200 × g for 10 min at RT, with the centrifuge brake on maximum settings.After centrifugation, 5 mL of plasma from the top layer of each sample was transferred to sterile 15-mL conical tubes and stored at −80˚C for later downstream analysis.The remaining upper plasma layer was discarded without disturbing the Lymphoprep-plasma interface.
PBMCs were collected from the Lymphoprep-plasma interface layer into 15-mL conical tubes, washed with up to 10 mL PBS-EDTA-FBS buffer, and centrifuged at 400 × g for 8 min at RT. PBMC pellets were resuspended in 1 mL RBC lysis buffer and incubated for 5 min at 37˚C to deplete contaminating RBCs from PBMCs.PBMCs were then washed once more after RBC lysis by adding up to 10 mL PBS-EDTA-FBS followed by centrifugation for 8 min at 400 × g at RT. PBMC pellets were resuspended in 0.5 mL of cell freezing media (90% FBS/ 10% DMSO), transferred to cryovials, and placed in a Mr. Frosty freezing container (Thermo Scientific, catalog #5100-0001) with isopropanol at −80˚C.After at least 12 h in the −80˚C freezer, PBMC-containing cryovials were transferred to a liquid nitrogen tank for long-term storage.For analysis, PBMCs were rapidly thawed in a 37˚C water bath with constant swirling, transferred to a 15-mL conical tube containing 8 mL of warm RPMI 1640 + 10% FBS media, recovered by centrifugation at 400 × g for 8 min, resuspended in 200 µL lysis buffer (PBS + 1% Triton X-100 + 1 Complete Mini protease inhibitor tablet), and kept on ice for 15 min.Lysates were centrifugated at 16,000 × g for 15 min at 4˚C to spin out cell debris, and the supernatant was collected for protein and nanoluciferase assays as described below.
RBC lysates were prepared by transferring 1 mL of RBC sample from the bottom of each SepMate-15 tube to a 15-mL conical tube.RBCs were lysed with 1 mL of RBC lysis buffer for 5 min at 37˚C, and RBC lysates were stored directly at −80˚C.

. Nanoluciferase assays
Nanoluciferase assays were used to quantify the presence of PalmGRET-EVs.50 µL undiluted plasma, RBC lysate, and PBMC lysate samples were loaded in duplicate into a 96-well white flat bottom polystyrene plate (Corning, catalog #3922).Nanoluciferase activity was measured using the Nano-Glo Luciferase Assay System (Promega, catalog #N1110) per the manufacturer's instructions.Briefly, Nano-Glo luciferase assay reagent was prepared just before use by combining one volume Nano-Glo luciferase assay substrate (furimazine) with 50 volumes Nano-Glo luciferase assay buffer.50 µL Nano-Glo luciferase assay reagent was added to wells containing samples, and nanoluciferase activity was measured immediately on a BioTek Synergy 2 microplate reader in luminescence mode, integration time 20 ms.Results were normalized by total protein concentrations as determined by Pierce BCA Protein Assay Kit (ThermoFisher Scientific, catalog #23225).PBMC lysates were not diluted for this assay; plasma and RBC lysates were diluted 12.5-fold in PBS.

. Statistical analysis
Experimental replicates are defined in the figure legends for each experiment.Flow cytometry data were analyzed with FlowJo software (v10.8).Statistical analyses of flow cytometry data were performed with GraphPad Prism 9.0 (GraphPad Software Inc.) using multiple comparison analysis testing in two-way analysis of variance (ANOVA) with Tukey's post-hoc test, or two-tailed paired t-tests, with p < 0.05 considered statistically significant.

. Availability of protocols
We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase and are available under the EV-TRACK ID: EV230584 (Van Deun et al., 2017).

 RESULTS
We used ex vivo whole blood to reproduce and extend our unexpected finding that intravenously administered EVs associate at high levels with B cells in primates (Driedonks et al., 2022).Our experimental design, using whole blood from donor subjects and F I G U R E  Schematic overview of the study design.Whole blood from pig-tailed macaques was drawn directly into a collection tube containing ACD.
Whole blood was transferred to microcentrifuge tubes for EV spike-in time course and dose-response assays.Tubes were removed from the rotating incubator at predefined time points, and 100 µL of blood was set aside for PBMC staining and flow cytometry analysis.The remaining blood was divided into plasma, red blood cell lysates, and PBMCs using density gradient centrifugation.Once separated, each whole blood component was stored as indicated until analysis by nanoluciferase assays.Created with BioRender.com.
maintained at 37˚C, matched the conditions of the in vivo study as much as possible (Figure 1).Expi293F-derived EVs that were introduced into blood were labelled genetically with the dual reporter palmitoylated EGFP-nanoluciferase protein (PalmGRET) (Wu et al., 2020) and/or post-production with the near-infrared, self-quenching membrane dye MemGlow 700 (Figure S1A).

. Characterization of MemGlow-PalmGRET EVs
MemGlow-PalmGRET EVs were characterized for particle concentration, size, and % GFP positivity (Figure S1B, C).By nanoflow cytometry analysis, EV preparations contained > 50% GFP+ particles, consistent with our previous results (Driedonks et al., 2022).22 two-fold serial dilutions of EVs, starting from a concentration of 2.0E+10 particles/mL, were measured by nanoluciferase assay to assess linear range (Figure S2A).Since correlation between particle input and relative light units (RLU) weakened below 6.5 RLU or 1.64E+06 particles/mL, we considered signal below 6.5 RLU to be background signal throughout this study (Figure S2B).Please note that the particle counts here, as established by NFCM in our laboratory, may not be comparable with particle counts obtained in other laboratories or with other methods (Welsh et al., 2023).

. Stability of EV-associated nanoluciferase in compartments of whole blood
We performed a 24-h time course to test the stability and distribution of nanoluciferase signal from EVs spiked into whole blood ex vivo.After fractionating blood treated with EVs or into plasma, RBC, and PBMC compartments, relative nanoluciferase activity (normalized by total protein concentration) remained detectable in each compartment for 24 h after spike-in (Figure 2).In contrast with our in vivo findings (Driedonks et al., 2022), nanoluciferase activity remained relatively stable over 24 h in plasma and PBMC compartments of ex vivo blood (Figure 2a, c, and d).Signal was comparatively low in the RBC compartment and declined steadily (Figure 2b).There was consistently more signal in plasma than PBMCs at each time point (Figure 2d), although not always statistically significant due to variability.

. Time course analysis of EV interactions with PBMC subtypes
To determine which populations of PBMCs interact with EVs in whole blood ex vivo, subsets of PBMCs in whole blood were labeled with a fluorescent antibody panel at 5 min, 1 h, 2 h, 4 h, or 24 h after spike-in of 8E+08 EVs or PBS.See Figures S3 and  S4 for cell and GFP+ and/or MemGlow+ gating.In line with our in vivo observations (Driedonks et al., 2022), EV signal was strongly associated with B cells across all time points (Figures 3a, 4a, and S4A).Around 80% of CD20 + B cells were positive for both genetic label and dye label at early time points, with signal declining slightly by 24 h (Figure 3a).Lower levels of signal were found in association with the other PBMC subtypes, and predominantly at early time points (Figure 3b-f; see also Figure 4b-f).
Figure S4 shows the distribution of GFP and MemGlow in the form of overlaid dot plots and histograms.Of potential interest, although MemGlow and GFP signals were concordant in CD20 + B cells, this was not the case for every PBMC subtype.For example, MemGlow signal in CD3 + T cells and CD3 + CD4 + T cells at 1 h was lower than GFP+ signal (Figure 3b,c), while the opposite was observed for CD159a NK cells and monocytes (Figure 3e,d).

. Effect of EV source and separation strategy on PBMC-EV interactions ex vivo
To this point, our in vivo and ex vivo results were obtained with Expi293F EVs that were separated using a standard workflow as previously published (Driedonks et al., 2022).To determine whether EVs from a different donor cell line, or obtained with a different separation protocol, would show a different distribution across blood compartments, we tested EVs from U-87 MG (glioma, "U-87") cell culture that were separated by two methods: differential ultracentrifugation (UC) and size exclusion chromatography (SEC) (Figure S5A).Standard characterization was done for these EV preparations (Théry et al., 2018).Mean particle size and concentration and EV markers were comparable (Figure S5B, C, D), but cellular marker calnexin was more pronounced in UC-separated EVs (Figure S5D).Similarly, bovine serum albumin, a common co-isolate from growth media, was detected in UC-but not SEC-separated EVs.U-87 EVs were labelled with MemGlow and ultrafiltrated to remove unbound dye.We note that particle concentrations were lower after UF, as previously reported (Rautaniemi et al., 2021;C. Zhou et al., 2023), and that the size distribution profile of MemGlow-U-87 EVs was narrower compared with unlabelled EVs (Figure S5E, F).U-87 EVs associated preferentially with CD20 + B cells at levels similar to those of Expi293F EVs (Figure 4a).Association of UC EVs was approximately 20% lower than for SEC EVs.Substantial variability for other cell types precludes firm conclusions except for the reproducible, much lower association of EV signal with non-B cell PBMCs (Figure 4b-f).Thus, EV from two donor cell types behaved similarly with regards to association with PBMC subtypes.

. Staining of PalmGRET-EV preparations with MemGlow alters EV GFP signal but not preferential associations between PalmGRET-EVs and B cells
Labelling of EVs with lipophilic dyes might change various properties of an EV preparation.The process of labelling with Mem-Glow appeared to decrease the percentage of PalmGRET EVs that were detected as positive for GFP (Figure S1C, E).Although this could be due to dye-mediated physical changes to the EVs, it might be explained most simply by formation of dye self-aggregates that decrease the EV share of the overall particle count.To understand if MemGlow labelling changed the cell association of EVs, 8E+08 PalmGRET EVs with or without MemGlow were tested in the ex vivo system.At 30 minutes, association was similar between the two populations of EVs and consistent with previous results (Figure S6).

. Influence of dosage on PalmGRET EV interactions with PBMC subtypes
Results of our previous time-course experiments suggest that EVs interact with PBMCs at levels detectable by flow cytometry as early as 5 min post-spike, peaking within 1 h.We thus chose 30 min as the time point to interrogate the possible effect of EV dosage on cell associations, exposing blood from four donors to 8E+08 (dose 1), 4E+09 (dose 2), and 1.8E+10 (dose 3) PalmGRET EVs without MemGlow.There was a dose-dependent increase in GFP+ signal for all PBMC subtypes (Figure 6a).At the highest EV dose, all queried PBMC subtypes were on average >1% GFP+ (Figure 6b-g).Importantly, monocytes disproportionately associated with EVs at the highest dose: on average, 26.2% of monocytes were GFP+ at dose #3 (Figure 6g).As a result, although CD20 + B cells were the predominant GFP+ cell type at dose #1 (as in previous experiments) (Figure 7a), the balance of GFP+ cells shifted to monocytes with increasing dose (Figure 7b).

 DISCUSSION
Complementary to our recent in vivo biodistribution study, the present study demonstrated that ex vivo stimulation of whole blood with fluorescently-and genetically-labelled EVs is a reliable, sensitive, and physiologically relevant model suitable to study blood cell interactions with EVs.Previously, we demonstrated that EVs could be detected in association with CD20 + B cells within 1 min of intravenous administration into the circulation of pig-tailed macaques.Remarkably, the ex vivo system used in this study also showed that EVs preferentially associate with CD20 + B cells in whole blood at levels similar to those detected in vivo in an EV dose-dependent manner.To the best of our knowledge, exposing whole blood ex vivo to labelled EVs as a means to quantify EV-PBMC interactions has not been performed previously.
(b) (a)   F I G U R E  PalmGRET EVs preferentially interact with CD20 + B cells in an EV dose-response relationship 30 min after exposure to whole blood ex vivo.Cell-associated GFP signal was quantified by flow cytometry after 30 min of whole blood exposure to three doses of PalmGRET EVs: 8E+08 (Dose 1), 4E+09 (Dose 2), and 1.8E+10 (Dose 3).Data are expressed as % GFP+ PBMCs with all cell types and donor replicates plotted together (a) or separately (b-g) for (b) CD20 + B cells (c) CD3 + T cells, (d) CD3 + CD4 + T cells, (e) CD3 + CD8 + T cells, (f) CD159a + NK cells, and (g).Curves in (a): arithmetic mean percentage of replicates (n = 4) and error bars: standard error of the mean.Connected symbols in (b-g) represent individual donors.Statistical comparisons for GFP+ CD20 + B cells and GFP+ monocytes at the same EV dose were performed using two-way ANOVA with Tukey's post-hoc test, **p < 0.01.
Previous work by Fendl and colleagues established that the pre-analytical parameters chosen for collection and storage of whole blood can induce post-sampling release of EVs (Fendl et al., 2016).Additionally, it has been well documented that different sample preparation methods can cause artifactual blood cell activation thereby altering cell phenotype, gene expression, and cell behaviour (Alharbi et al., 2019;Weber et al., 2018).Because experimental parameters likely influence how blood cells interact with EVs, the anticoagulant type, sample volume, handling, storage, transport, and incubation conditions should be carefully selected and compared.Blood cells are physically compacted in a confined space in vivo and changes to their environment can alter their cellular state and by extension interactions with neighbouring cells and small particles, such as EVs.Shear force, blood flow, oxygen tension, three-dimensional architectural arrangement, and the presence of endothelial cells are parameters that are more technically challenging to recreate ex vivo but should also be considered, as they have been shown to influence the dynamics of blood cells in circulation in vivo (Campinho et al., 2020;Mitchell et al., 2023).A potential strategy that could be explored in the future to examine the interaction network between EVs and blood cells involves the use of microfluidic devices.Due to recent advances in microfluidic technology which allow for tight control of system inputs, microfluidic devices can be customized to provide a continuous supply of oxygen, shear rate, and flow to blood samples to better represent physiological conditions (Y.J. Kang, 2022;Kang & Lee, 2018;Sebastian & Dittrich, 2018).A comparison of these variables and techniques are warranted to inform future studies.
It was recently reported that labelling of PalmGRET-EVs with lipophilic dyes PKH26, DiD, and DiR caused an increase in EV size compared to their unlabelled EV counterparts (Magoling et al., 2023).Changes in EV size are not inconsequential in biodistribution studies given that EV trafficking and uptake by recipient cells are partially determined by particle size (Caponnetto et al., 2017;Pedrioli & Paganetti, 2021).Here, we did not observe an increase in the mean size of MemGlow-PalmGRET EVs compared with PalmGRET-EVs.However, nano-flow cytometry analysis showed that MemGlow labelling of PalmGRET EVs decreased the % of GFP+ particles in PalmGRET EV preparations.It is unclear why MemGlow labelling would lead to a reduced percentage of GFP+ particles.The PalmGRET reporter anchors to the inner membrane leaflet of EVs (Magoling et al., 2023;Wu et al., 2020), and the short, 12-carbon fatty acid tails of the MemGlow dyes (Collot et al., 2019) may label membranes without disturbing the inner membrane leaflet (Skotland & Sandvig, 2019).Possibly, dye aggregates or dye interactions with non-EV substances may add to the total number of particles in the population.Nonetheless, no substantial differences were detected in terms of % GFP+ PBMC subsets when we compared the pattern of interactions between PBMCs and PalmGRET EVs labelled with or without MemGlow after co-incubation in whole blood ex vivo for 30 min at an EV dose of 8E+08.This suggests that both PalmGRET and MemGlow can be used reliably, either in combination or separately, to enumerate the interactions between blood cells and EVs ex vivo.
In the present study we detected a comparable degree of interactions between CD20 + B cells and EVs separated from the conditioned media of Expi293F-PalmGRET and U-87 cells.Expi293F-PalmGRET EVs and U-87 EVs isolated by SEC did not differ in terms of their preferential targeting of B cells.Moreover, U-87 EVs interacted at similar levels with CD20 + B cells, independent of the method of EV isolation.EVs produced by different cell types, including immune cells, stem cells, astrocytes, and cancer cells, often exhibit distinct biophysical properties and functions that typically align with those ascribed to their parental cells at the time of EV release (Veerman et al., 2019;S. Zhao et al., 2021).Nevertheless, EVs from two very different cell sources interacted with apparent preference with CD20 + B cells.It remains to be seen if this tropism is maintained by EVs from other cell types.
Our studies in vivo and ex vivo suggest that, at least for Expi293F and U-87 EVs, surface engineering of EVs may be dispensable for B-cell tropism in blood.Cellular tropism of EVs, including mechanisms of EV interactions with B cells, is an emerging area of interest (Limongi et al., 2021;Oshchepkova et al., 2021).Previously, EVs have been surface-engineered with the major envelope glycoprotein of Epstein-Barr Virus (EBV), gp350, known to mediate attachment of EBV to B cells.On EVs, gp350 conferred B-cell tropism and cargo delivery functionality both in vitro (Vallhov et al., 1950;Xiu et al., 2022) and in vivo (Xiu et al., 2022).Platelet-derived EVs have also been shown to stimulate the production of antibodies from immortalized and primary B cells in vitro, although EV-B cell interactions were not specifically assayed (Khayati & Yari, 2019;Yari et al., 2018).Microparticles derived from Kato cells interacted at high levels with B cells in vitro when the microparticles were preincubated with complementcontaining human serum (Köppler et al., 2006).Altogether, our studies and these previous findings support the notion that non-surface-engineered EVs may interact preferentially with B cells due to factors present in whole or fractionated blood.That is, preferential interactions with B cells occur primarily when EVs are isolated from blood samples, pre-incubated with blood serum, or added directly into whole blood samples ex vivo.Emerging evidence shows that when nano-sized particles, including EVs, are introduced into blood, they become coated with a "corona" (Hadjidemetriou et al., 2019;Wolf et al., 2022) of proteins and other molecules that may influence particle-cell interactions.Possibly, non-surface-engineered EVs must adsorb plasma components to mediate interactions with B cells.
Although we found that B cells were the dominant PBMC subtype that interacted with EVs upon exposure to a low EV dose, interactions between EVs and monocytes at higher EV doses accounted for > 50% of the cumulative GFP signal in CD3 + T cells, CD20 + B cells, and monocytes (Figure 7b).It is unclear if the dose-dependent shift in EV associations with PBMCs could simply be a result of enhanced recognition of EVs by phagocytic cells at high EV dosage or if it is due to a higher presence of phagocytic cells for clearance of EVs (Gustafson et al., 2015;Parada et al., 2021).Another possibility is that EVs saturate a specific receptor or receptors on CD20 + B cells in blood at a lower dose, leaving more unbound EVs to interact with other cell types such as monocytes.Learning more about the links between EV dosage and uptake, processing, and clearance mechanisms by phagocytic cells in blood will provide a basis to maximize EV circulation time and uptake by target cells for future clinical applications.
B cells constitute a vital component of the adaptive humoral immune system.They are generally categorized as naïve, activated, memory, or plasma cells (Allman & Pillai, 2008;Maecker et al., 2012).B cell subsets are largely defined by the relative expression of specific cell surface markers that correspond to different stages of B cell development, maturation, and activation.Naïve B cells express CD19 and CD24 and lack CD27.Upon activation by antigens, resting naïve B cells express CD80 and CD86 and gain the ability to differentiate into either plasma cells or memory B cells.Plasma cells express high levels of CD138 and CD38 while exhibiting low or no expression of the B-cell lineage markers CD19 and CD20.Memory B cells, on the other hand, are distinguished by the presence of CD19, CD20, and CD27 and the absence of CD138.CD19 is consistently expressed across all stages of B cell differentiation, except for plasma cells, thereby making it a reliable marker for B cell detection.In the context of this study, B cells are defined as CD3-CD20+ cells.Note that CD20 was used to phenotype macaque B cells, substituting the conventional human B cell marker, CD19 (Gujer et al., 2011).We are currently investigating whether EVs interact differentially with different B cell subsets.
Since the flow cytometry methods we used here do not identify subcellular localization, our results are agnostic to the nature of EV-B cell association, that is, surface interaction or internalization.We are currently addressing the subcellular fate of PBMCand B cell-associated EVs in whole blood and whether these EVs influence cell functionality.However, both surface interaction and internalization are consistent with EV functions.Whereas internalization and membrane fusion are needed for cytoplasmic payload delivery, EVs may also communicate via surface-surface interactions that trip intracellular signalling pathways, thereby modulating recipient cell functionality.
Ex vivo models are useful to study EV interactions and functions in complex biological environments that are considered more physiologically relevant than simplified, monocultured single cell lines as in vitro models.Overall, this work supports the use of an ex vivo whole blood platform to interrogate PBMC-EV interactions in blood, which could help better understand the fate of intravenously injected EVs at the cellular and molecular level.While we limited this study to non-human primate whole blood, this ex vivo system could be easily tailored to study EV interactions with blood cells from other species.We fully expect this ex vivo model to serve as an intermediate step to investigate a broad range of physiological questions regarding the distribution and pharmacokinetics of labelled EVs in blood, while simultaneously eliminating the time, animal, and monetary costs of in vivo pharmacokinetics and biodistribution studies.

AU T H O R C O N T R I B U T I O N S
Yi Wen: Data curation; Formal analysis; Investigation; Methodology; Visualization.Samuel Vazquez: Formal analysis; Investigation; Methodology.Olesia Gololobova: Formal analysis; Investigation; Methodology.Amanda Maxwell: Methodology; Project administration.Jessica Plunkard: Methodology; Project administration.Natalie Castell: Methodology.Bess Carlson: Methodology.

A C K N O W L E D G E M E N T S
The authors thank members of the Witwer Laboratory, the Retrovirus Laboratory, and Research Animal Resources for helpful discussions and suggestions.This research project was supported by the US National Institutes of Health through AI144997 (to KWW) and U42OD013117 (to EK Hutchinson).The Witwer lab is also supported in part by NCI/Common Fund CA241694, NIMH MH118164, and the Richman Family Precision Medicine Center of Excellence in Alzheimer's Disease at Johns Hopkins University.

C O N F L I C T S O F I N T E R E S T
KWW is or has been an advisory board member of ShiftBio, Exopharm, NeuroDex, NovaDip, and ReNeuron; holds stock options with NeuroDex; and privately consults as Kenneth Witwer Consulting.Ionis Pharmaceuticals, Yuvan Research, and AgriSciX have sponsored or are sponsoring research in the Witwer laboratory.
Distribution of EVs in macaque whole blood compartments at different time points ex vivo.PalmGRET EVs were detected by nanoluciferase assay in (a) plasma, (b) RBCs, and (c) PBMCs after addition of 8E+08 EVs or PBS.Nanoluciferase activity was normalized by protein concentration (BCA).Curves: mean of replicates (n = 3 or n = 2 donors as indicated for 2 h).(d) Comparison of nanoluciferase activity in plasma and PBMC compartments, normalized by total protein concentration.Each line represents a separate donor.Data points: results of two or three independent experiments, performed with two or three independent donors.Two-tailed paired t-tests were used to compare plasma and PBMC samples in D (p < 0.05 was considered significant).

F
I G U R E  PalmGRET EVs preferentially interact with CD20 + B cells over a 24 h time course.GFP and MemGlow signals were detected by flow cytometry for (a) CD20 + B cells (b) CD3 + T cells, (c) CD3 + CD4 + T cells, (d) CD3 + CD8 + T cells, (e) CD159a + NK cells, and (f) monocytes at time points over up to 24 h after spiking 8E+08 MemGlow-PalmGRET EVs into whole blood.Data are expressed as % GFP+ or %MemGlow+ for each PBMC subtype.Data are from experiments with blood from n = 2 donors.
MemGlow-labelled EVs from U-87 MG cells preferentially interact with B cells ex vivo.Quantification of data obtained by flow cytometry using the same workflow described for Figure3.Whole-blood was spiked with PBS MemGlow control, U-87 MG EVs labelled with MemGlow, or MemGlow-PalmGRET EVs from Expi293F culture and incubated for 5 min at 37˚C.Percentage of MemGlow+ PBMC subtypes were determined with flow cytometry.The bars represent the mean of two independent experiments, performed with samples from two independent donors.

F
Specific subsets of PBMCs and their contribution to EGFP+ PBMCs.(a) Comparison of PBMC subtype proportions and proportion of (b) GFP+ and (c) MemGlow 700 PBMC subsets (CD3 + T cells, CD20 + B cells, monocytes).Graphs display mean frequency as a percentage of total PBMCs gated by PBMC subsets, GFP, or MemGlow 700 fluorescence.Data are from experiments with blood from n = 2 donors.B cells are a relatively small population of total PBMCs but are the majority of detectable GFP+ and MemGlow+ PBMCs.
The dominant percentage of detectable EGFP+ PBMCs in PalmGRET-treated whole blood at different PalmGRET EV doses.(a) Comparison of PBMC subtype proportions and (b) proportion of GFP+ PBMC subsets (CD3 + T cells, CD20 + B cells, and monocytes).Graphs display mean frequency as a percentage of total PBMCs gated by PBMC subsets or GFP fluorescence.Data from experiments with blood of n = 4 donors.